energetics of biological macromolecules, part e

Nucleic Acids and Protein Synthesis Part C Edited by Kivie Moldave and Lawrence Grossman Volume XXI.. Nucleic Acids and Protein Synthesis Part E Edited by Lawrence Grossman and Kivie Mol

John N Abelson Melvin I Simon

DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY

PASADENA, CALIFORNIA

FOUNDING EDITORS

Sidney P Colowick and Nathan O Kaplan

Article numbers are in parentheses and following the names of contributors.

Affiliations listed are current.

Vahe Bandarian (7), Department of

Biochemistry, University of Arizona,

Tucson, Arizona 85721

James G Bann (18), Department of

Bio-chemistry and Molecular Biophysics,

Washington University School of

Medi-cine, St Louis, Missouri 63110

George Barany (17), Department of

Chemistry, University of Minnesota,

Minneapolis, Minnesota 55455

Elisar Barbar (11), Department of

Chem-istry and BiochemChem-istry, Ohio University,

Athens, Ohio 45701

Michael Carey (10), Department of

Bio-logical Chemistry, UCLA School of

Medicine, Los Angeles, California 90095

Nata`lia Carulla (17), Department of

Chemistry, Cambridge University,

Cam-bridge CB2 1EW, England

Hue Sun Chan (16), Department of

Bio-chemistry, University of Toronto,

Tor-onto, Ontario M5S 1A8, Canada

Eefie Chen (14), Department of

Chemis-try and BiochemisChemis-try, University of

Ca-lifornia, Santa Cruz, California 95064

Diana Chinchilla (4), CARB/University

of Maryland Biotechnology Institute,

Rockville, Maryland 20850

Edward Eisenstein (4),

CARB/Univer-sity of Maryland Biotechnology Institute,

Rockville, Maryland 20850

Carolyn A Fitch (2), Department of

Biophysics, Johns Hopkins University,

Baltimore, Maryland 21218

Carl Frieden (18), Department of chemistry and Molecular Biophysics, Washington University School of Medi- cine, St Louis, Missouri 63110

Bio-D Travis Gallagher (4), Biotech sion, Chemical Science and Technology Lab, National Institute of Standards and Technology, Gaithersburg, Maryland 20899

Divi-Bertrand Garci´a-Moreno E (2), partment of Biophysics, Johns Hopkins University, Baltimore, Maryland 21218 Robert A Goldbeck (14), Department of Chemistry and Biochemistry, University

De-of California, Santa Cruz, California 95064

Gregory A Grant (5), Department of Molecular Biology and Pharmacology, Washington University School of Medi- cine, St Louis, Missouri 63110

Michael Hare (11), Department of istry and Biochemistry, Ohio University, Athens, Ohio 45701

Chem-Sydney D Hoeltzli (18), Department of Biochemistry and Molecular Biophysics, Washington University School of Medi- cine, St Louis, Missouri 63110

Vasanthi Jayaraman (8), Department of Integrative Biology and Pharmacology, University of Texas Health Sciences Center, Houston, Texas 77030

Kristina M Johnson (10), Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90095

ix

Hu¨seyin Kaya (16), Department of

Biochemistry, University of Toronto,

Toronto, Ontario M5S 1A8, Canada

David S Kliger (14), Department of

Chemistry and Biochemistry, University

of California, Santa Cruz, California

95064

Heidi Lau (4), CARB/University of

Mary-land Biotechnology Institute, Rockville,

Maryland 20850

Susan Marqusee (15), Department of

Molecular and Cell Biology, University

of California, Berkeley, Berkeley,

Cali-fornia 94720

Rowena G Matthews (7), Biophysics

Research Division, University of

Michi-gan, Ann Arbor, Michigan 48109

Hai Pan (13), Amgen Inc., Thousand

Oaks, California 91320

Gregory D Reinhart (9), Department of

Biochemistry and Biophysics, Texas

A&M University, College Station, Texas

77843

Claudia N Schutz (3), Department of

Chemistry, University of Southern

Cali-fornia, Los Angeles, California 90089

Alan Senior (6), Department of

Biochem-istry and Biophysics, University of

Rochester Medical Center, Rochester,

New York 14642

Seishi Shimizu (16), Department of

Bio-chemistry, University of Toronto,

Tor-onto, Ontario M5S 1A8, Canada

Avital Shurki (3), Department of

Chem-istry, University of Southern California,

Los Angeles, California 90089

Andrea Smallwood (10), Department

of Biological Chemistry, UCLA School

of Medicine, Los Angeles, California

90095

David L Smith (13), Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588

Elaine Stephens (1), Department of Chemistry, University of Cambridge, Cambridge, CB2 1EW, England Marek Sˇtrajbl (3), Department of Chem- istry, University of Southern California, Los Angeles, California 90089

Jin Wang (10), Department of try, Ninjing University, Ninjing, People’s Republic of China

Biochemis-Arieh Warshel (3), Department of Chemistry, University of Southern Cali- fornia, Los Angeles, California 90089 Joachim Weber (6), Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lub- bock, Texas 79430

David Wildes (15), Department of cular and Cell Biology, University of California, Berkeley, Berkeley, Califor- nia 94720

Mole-Dudley H Williams (1), Department of Chemistry, University of Cambridge, Cambridge, CB2 1EW, England Clare Woodward (17), Department of Biochemistry, Biophysics and Molecular Biology, University of Minnesota, St Paul, Minnesota 55108

Robert W Woody (12), Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, Colorado 80525

Rosa Zerella (1), Department of istry, University of Cambridge, Cam- bridge, CB2 1EW, England

Chem-Min Zhou (1), Department of Chemistry, University of Cambridge, Cambridge, CB2 1EW, England

One of the most intriguing problems in biological energetics is that of tivity From the discovery of cooperativity and allostery in hemoglobin 100years ago (Bohr et al., 1904)1 to the characterization of cooperativity in amyriad of processes in modern times (i.e., transport, catalysis, signaling, assem-bly, folding), the molecular mechanisms by which energy is transferred fromone part of a macromolecule to another continues to challenge us Of course,the problem has many layers, as a molecule as ‘‘simple’’ and familiar ashemoglobin can simultaneously sense the chemical potential of each physiolo-gical ligand and adjust its interactions with the others accordingly Ironically,the very allosteric intermediates that hold the structural and energetic secrets

coopera-of cooperativity are the same whose populations are suppressed and, in manyinstances, largely obscured by the nature of cooperativity itself Thus, innova-tive methodologies and techniques have been developed to address coopera-tive systems, many of which are presented in this volume Energetics ofBiological Macromolecules Part E and its companion volume, Part D Thereader will observe remarkable similarities among the wide range of experi-mental strategies employed, attesting to fundamental issues inherent in allcooperative systems

Jo M HoltMichael L JohnsonGary K Ackers

1 C Bohr, K A Hasselbach, and A Krogh, Skand Arch Physiol 16, 402 (1904).

xi

METHODS IN ENZYMOLOGY

Volume I Preparation and Assay of Enzymes

Edited by Sidney P Colowick and Nathan O Kaplan

Volume II Preparation and Assay of Enzymes

Edited by Sidney P Colowick and Nathan O Kaplan

Volume III Preparation and Assay of Substrates

Edited by Sidney P Colowick and Nathan O Kaplan

Volume IV Special Techniques for the Enzymologist

Edited by Sidney P Colowick and Nathan O Kaplan

Volume V Preparation and Assay of Enzymes

Edited by Sidney P Colowick and Nathan O Kaplan

Volume VI Preparation and Assay of Enzymes (Continued)

Preparation and Assay of Substrates

Special Techniques

Edited by Sidney P Colowick and Nathan O Kaplan

Volume VII Cumulative Subject Index

Edited by Sidney P Colowick and Nathan O Kaplan

Volume VIII Complex Carbohydrates

Edited by Elizabeth F Neufeld and Victor Ginsburg

Volume IX Carbohydrate Metabolism

Edited by Willis A Wood

Volume X Oxidation and Phosphorylation

Edited by Ronald W Estabrook and Maynard E Pullman

Volume XI Enzyme Structure

Edited by C H W Hirs

Volume XII Nucleic Acids (Parts A and B)

Edited by Lawrence Grossman and Kivie Moldave

Volume XIII Citric Acid Cycle

Edited by J M Lowenstein

Volume XIV Lipids

Edited by J M Lowenstein

Volume XV Steroids and Terpenoids

Edited by Raymond B Clayton

xiii

Volume XVI Fast Reactions

Edited by Kenneth Kustin

Volume XVII Metabolism of Amino Acids and Amines (Parts A and B)Edited by Herbert Tabor and Celia White Tabor

Volume XVIII Vitamins and Coenzymes (Parts A, B, and C)

Edited by Donald B McCormick and Lemuel D Wright

Volume XIX Proteolytic Enzymes

Edited by Gertrude E Perlmann and Laszlo Lorand

Volume XX Nucleic Acids and Protein Synthesis (Part C)

Edited by Kivie Moldave and Lawrence Grossman

Volume XXI Nucleic Acids (Part D)

Edited by Lawrence Grossman and Kivie Moldave

Volume XXII Enzyme Purification and Related Techniques

Edited by William B Jakoby

Volume XXIII Photosynthesis (Part A)

Edited by Anthony San Pietro

Volume XXIV Photosynthesis and Nitrogen Fixation (Part B)

Edited by Anthony San Pietro

Volume XXV Enzyme Structure (Part B)

Edited by C H W Hirs and Serge N Timasheff

Volume XXVI Enzyme Structure (Part C)

Edited by C H W Hirs and Serge N Timasheff

Volume XXVII Enzyme Structure (Part D)

Edited by C H W Hirs and Serge N Timasheff

Volume XXVIII Complex Carbohydrates (Part B)

Edited by Victor Ginsburg

Volume XXIX Nucleic Acids and Protein Synthesis (Part E)

Edited by Lawrence Grossman and Kivie Moldave

Volume XXX Nucleic Acids and Protein Synthesis (Part F)

Edited by Kivie Moldave and Lawrence Grossman

Volume XXXI Biomembranes (Part A)

Edited by Sidney Fleischer and Lester Packer

Volume XXXII Biomembranes (Part B)

Edited by Sidney Fleischer and Lester Packer

Volume XXXIII Cumulative Subject Index Volumes I-XXX

Edited by Martha G Dennis and Edward A Dennis

Volume XXXIV Affinity Techniques (Enzyme Purification: Part B)Edited by William B Jakoby and Meir Wilchek

Volume XXXV Lipids (Part B)

Edited by John M Lowenstein

Volume XXXVI Hormone Action (Part A: Steroid Hormones)

Edited by Bert W O’Malley and Joel G Hardman

Volume XXXVII Hormone Action (Part B: Peptide Hormones)

Edited by Bert W O’Malley and Joel G Hardman

Volume XXXVIII Hormone Action (Part C: Cyclic Nucleotides)

Edited by Joel G Hardman and Bert W O’Malley

Volume XXXIX Hormone Action (Part D: Isolated Cells, Tissues, and OrganSystems)

Edited by Joel G Hardman and Bert W O’Malley

Volume XL Hormone Action (Part E: Nuclear Structure and Function)Edited by Bert W O’Malley and Joel G Hardman

Volume XLI Carbohydrate Metabolism (Part B)

Edited by W A Wood

Volume XLII Carbohydrate Metabolism (Part C)

Edited by W A Wood

Volume XLIII Antibiotics

Edited by John H Hash

Volume XLIV Immobilized Enzymes

Edited by Klaus Mosbach

Volume XLV Proteolytic Enzymes (Part B)

Edited by Laszlo Lorand

Volume XLVI Affinity Labeling

Edited by William B Jakoby and Meir Wilchek

Volume XLVII Enzyme Structure (Part E)

Edited by C H W Hirs and Serge N Timasheff

Volume XLVIII Enzyme Structure (Part F)

Edited by C H W Hirs and Serge N Timasheff

Volume XLIX Enzyme Structure (Part G)

Edited by C H W Hirs and Serge N Timasheff

Volume L Complex Carbohydrates (Part C)

Edited by Victor Ginsburg

Volume LI Purine and Pyrimidine Nucleotide Metabolism

Edited by Patricia A Hoffee and Mary Ellen Jones

Volume LII Biomembranes (Part C: Biological Oxidations)

Edited by Sidney Fleischer and Lester Packer

Volume LIII Biomembranes (Part D: Biological Oxidations)

Edited by Sidney Fleischer and Lester Packer

Volume LIV Biomembranes (Part E: Biological Oxidations)

Edited by Sidney Fleischer and Lester Packer

Volume LV Biomembranes (Part F: Bioenergetics)

Edited by Sidney Fleischer and Lester Packer

Volume LVI Biomembranes (Part G: Bioenergetics)

Edited by Sidney Fleischer and Lester Packer

Volume LVII Bioluminescence and Chemiluminescence

Edited by Marlene A DeLuca

Volume LVIII Cell Culture

Edited by William B Jakoby and Ira Pastan

Volume LIX Nucleic Acids and Protein Synthesis (Part G)

Edited by Kivie Moldave and Lawrence Grossman

Volume LX Nucleic Acids and Protein Synthesis (Part H)

Edited by Kivie Moldave and Lawrence Grossman

Volume 61 Enzyme Structure (Part H)

Edited by C H W Hirs and Serge N Timasheff

Volume 62 Vitamins and Coenzymes (Part D)

Edited by Donald B McCormick and Lemuel D Wright

Volume 63 Enzyme Kinetics and Mechanism (Part A: Initial Rate andInhibitor Methods)

Edited by Daniel L Purich

Volume 64 Enzyme Kinetics and Mechanism (Part B: Isotopic Probes andComplex Enzyme Systems)

Edited by Daniel L Purich

Volume 65 Nucleic Acids (Part I)

Edited by Lawrence Grossman and Kivie Moldave

Volume 66 Vitamins and Coenzymes (Part E)

Edited by Donald B McCormick and Lemuel D Wright

Volume 67 Vitamins and Coenzymes (Part F)

Edited by Donald B McCormick and Lemuel D Wright

Volume 68 Recombinant DNA

Edited by Ray Wu

Volume 69 Photosynthesis and Nitrogen Fixation (Part C)

Edited by Anthony San Pietro

Volume 70 Immunochemical Techniques (Part A)

Edited by Helen Van Vunakis and John J Langone

Volume 71 Lipids (Part C)

Edited by John M Lowenstein

Volume 72 Lipids (Part D)

Edited by John M Lowenstein

Volume 73 Immunochemical Techniques (Part B)

Edited by John J Langone and Helen Van Vunakis

Volume 74 Immunochemical Techniques (Part C)

Edited by John J Langone and Helen Van Vunakis

Volume 75 Cumulative Subject Index Volumes XXXI, XXXII, XXXIV–LXEdited by Edward A Dennis and Martha G Dennis

Volume 76 Hemoglobins

Edited by Eraldo Antonini, Luigi Rossi-Bernardi, and Emilia ChianconeVolume 77 Detoxication and Drug Metabolism

Edited by William B Jakoby

Volume 78 Interferons (Part A)

Edited by Sidney Pestka

Volume 79 Interferons (Part B)

Edited by Sidney Pestka

Volume 80 Proteolytic Enzymes (Part C)

Edited by Laszlo Lorand

Volume 81 Biomembranes (Part H: Visual Pigments and Purple Membranes, I)Edited by Lester Packer

Volume 82 Structural and Contractile Proteins (Part A: Extracellular Matrix)Edited by Leon W Cunningham and Dixie W Frederiksen

Volume 83 Complex Carbohydrates (Part D)

Edited by Victor Ginsburg

Volume 84 Immunochemical Techniques (Part D: Selected Immunoassays)Edited by John J Langone and Helen Van Vunakis

Volume 85 Structural and Contractile Proteins (Part B: The ContractileApparatus and the Cytoskeleton)

Edited by Dixie W Frederiksen and Leon W Cunningham

Volume 86 Prostaglandins and Arachidonate Metabolites

Edited by William E M Lands and William L Smith

Volume 87 Enzyme Kinetics and Mechanism (Part C: Intermediates,

Stereo-chemistry, and Rate Studies)

Edited by Daniel L Purich

Volume 88 Biomembranes (Part I: Visual Pigments and Purple Membranes, II)Edited by Lester Packer

Volume 89 Carbohydrate Metabolism (Part D)

Edited by Willis A Wood

Volume 90 Carbohydrate Metabolism (Part E)

Edited by Willis A Wood

Volume 91 Enzyme Structure (Part I)

Edited by C H W Hirs and Serge N Timasheff

Volume 92 Immunochemical Techniques (Part E: Monoclonal Antibodies andGeneral Immunoassay Methods)

Edited by John J Langone and Helen Van Vunakis

Volume 93 Immunochemical Techniques (Part F: Conventional Antibodies,

Fc Receptors, and Cytotoxicity)

Edited by John J Langone and Helen Van Vunakis

Volume 94 Polyamines

Edited by Herbert Tabor and Celia White Tabor

Volume 95 Cumulative Subject Index Volumes 61–74, 76–80

Edited by Edward A Dennis and Martha G Dennis

Volume 96 Biomembranes [Part J: Membrane Biogenesis: Assembly andTargeting (General Methods; Eukaryotes)]

Edited by Sidney Fleischer and Becca Fleischer

Volume 97 Biomembranes [Part K: Membrane Biogenesis: Assembly andTargeting (Prokaryotes, Mitochondria, and Chloroplasts)]

Edited by Sidney Fleischer and Becca Fleischer

Volume 98 Biomembranes (Part L: Membrane Biogenesis: Processing andRecycling)

Edited by Sidney Fleischer and Becca Fleischer

Volume 99 Hormone Action (Part F: Protein Kinases)

Edited by Jackie D Corbin and Joel G Hardman

Volume 100 Recombinant DNA (Part B)

Edited by Ray Wu, Lawrence Grossman, and Kivie Moldave

Volume 101 Recombinant DNA (Part C)

Edited by Ray Wu, Lawrence Grossman, and Kivie Moldave

Volume 102 Hormone Action (Part G: Calmodulin and Calcium-BindingProteins)

Edited by Anthony R Means and Bert W O’Malley

Volume 103 Hormone Action (Part H: Neuroendocrine Peptides)

Edited by P Michael Conn

Volume 104 Enzyme Purification and Related Techniques (Part C)

Edited by William B Jakoby

Volume 105 Oxygen Radicals in Biological Systems

Edited by Lester Packer

Volume 106 Posttranslational Modifications (Part A)

Edited by Finn Wold and Kivie Moldave

Volume 107 Posttranslational Modifications (Part B)

Edited by Finn Wold and Kivie Moldave

Volume 108 Immunochemical Techniques (Part G: Separation and

Characterization of Lymphoid Cells)

Edited by Giovanni Di Sabato, John J Langone, and Helen Van VunakisVolume 109 Hormone Action (Part I: Peptide Hormones)

Edited by Lutz Birnbaumer and Bert W O’Malley

Volume 110 Steroids and Isoprenoids (Part A)

Edited by John H Law and Hans C Rilling

Volume 111 Steroids and Isoprenoids (Part B)

Edited by John H Law and Hans C Rilling

Volume 112 Drug and Enzyme Targeting (Part A)

Edited by Kenneth J Widder and Ralph Green

Volume 113 Glutamate, Glutamine, Glutathione, and Related CompoundsEdited by Alton Meister

Volume 114 Diffraction Methods for Biological Macromolecules (Part A)Edited by Harold W Wyckoff, C H W Hirs, and Serge N TimasheffVolume 115 Diffraction Methods for Biological Macromolecules (Part B)Edited by Harold W Wyckoff, C H W Hirs, and Serge N TimasheffVolume 116 Immunochemical Techniques (Part H: Effectors and Mediators ofLymphoid Cell Functions)

Edited by Giovanni Di Sabato, John J Langone, and Helen Van VunakisVolume 117 Enzyme Structure (Part J)

Edited by C H W Hirs and Serge N Timasheff

Volume 118 Plant Molecular Biology

Edited by Arthur Weissbach and Herbert Weissbach

Volume 119 Interferons (Part C)

Edited by Sidney Pestka

Volume 120 Cumulative Subject Index Volumes 81–94, 96–101

Volume 121 Immunochemical Techniques (Part I: Hybridoma Technologyand Monoclonal Antibodies)

Edited by John J Langone and Helen Van Vunakis

Volume 122 Vitamins and Coenzymes (Part G)

Edited by Frank Chytil and Donald B McCormick

Volume 123 Vitamins and Coenzymes (Part H)

Edited by Frank Chytil and Donald B McCormick

Volume 124 Hormone Action (Part J: Neuroendocrine Peptides)

Edited by P Michael Conn

Volume 125 Biomembranes (Part M: Transport in Bacteria, Mitochondria,and Chloroplasts: General Approaches and Transport Systems)

Edited by Sidney Fleischer and Becca Fleischer

Volume 126 Biomembranes (Part N: Transport in Bacteria, Mitochondria, andChloroplasts: Protonmotive Force)

Edited by Sidney Fleischer and Becca Fleischer

Volume 127 Biomembranes (Part O: Protons and Water: Structure andTranslocation)

Edited by Lester Packer

Volume 128 Plasma Lipoproteins (Part A: Preparation, Structure, andMolecular Biology)

Edited by Jere P Segrest and John J Albers

Volume 129 Plasma Lipoproteins (Part B: Characterization, Cell Biology, andMetabolism)

Edited by John J Albers and Jere P Segrest

Volume 130 Enzyme Structure (Part K)

Edited by C H W Hirs and Serge N Timasheff

Volume 131 Enzyme Structure (Part L)

Edited by C H W Hirs and Serge N Timasheff

Volume 132 Immunochemical Techniques (Part J: Phagocytosis and

Cell-Mediated Cytotoxicity)

Edited by Giovanni Di Sabato and Johannes Everse

Volume 133 Bioluminescence and Chemiluminescence (Part B)

Edited by Marlene DeLuca and William D McElroy

Volume 134 Structural and Contractile Proteins (Part C: The ContractileApparatus and the Cytoskeleton)

Edited by Richard B Vallee

Volume 135 Immobilized Enzymes and Cells (Part B)

Edited by Klaus Mosbach

Volume 136 Immobilized Enzymes and Cells (Part C)

Edited by Klaus Mosbach

Volume 137 Immobilized Enzymes and Cells (Part D)

Edited by Klaus Mosbach

Volume 138 Complex Carbohydrates (Part E)

Edited by Victor Ginsburg

Volume 139 Cellular Regulators (Part A: Calcium- and Calmodulin-BindingProteins)

Edited by Anthony R Means and P Michael Conn

Volume 140 Cumulative Subject Index Volumes 102–119, 121–134

Volume 141 Cellular Regulators (Part B: Calcium and Lipids)

Edited by P Michael Conn and Anthony R Means

Volume 142 Metabolism of Aromatic Amino Acids and Amines

Edited by Seymour Kaufman

Volume 143 Sulfur and Sulfur Amino Acids

Edited by William B Jakoby and Owen Griffith

Volume 144 Structural and Contractile Proteins (Part D: Extracellular Matrix)Edited by Leon W Cunningham

Volume 145 Structural and Contractile Proteins (Part E: Extracellular Matrix)Edited by Leon W Cunningham

Volume 146 Peptide Growth Factors (Part A)

Edited by David Barnes and David A Sirbasku

Volume 147 Peptide Growth Factors (Part B)

Edited by David Barnes and David A Sirbasku

Volume 148 Plant Cell Membranes

Edited by Lester Packer and Roland Douce

Volume 149 Drug and Enzyme Targeting (Part B)

Edited by Ralph Green and Kenneth J Widder

Volume 150 Immunochemical Techniques (Part K: In Vitro Models of B and TCell Functions and Lymphoid Cell Receptors)

Edited by Giovanni Di Sabato

Volume 151 Molecular Genetics of Mammalian Cells

Edited by Michael M Gottesman

Volume 152 Guide to Molecular Cloning Techniques

Edited by Shelby L Berger and Alan R Kimmel

Volume 153 Recombinant DNA (Part D)

Edited by Ray Wu and Lawrence Grossman

Volume 154 Recombinant DNA (Part E)

Edited by Ray Wu and Lawrence Grossman

Volume 155 Recombinant DNA (Part F)

Edited by Ray Wu

Volume 156 Biomembranes (Part P: ATP-Driven Pumps and RelatedTransport: The Na, K-Pump)

Edited by Sidney Fleischer and Becca Fleischer

Volume 157 Biomembranes (Part Q: ATP-Driven Pumps and RelatedTransport: Calcium, Proton, and Potassium Pumps)

Edited by Sidney Fleischer and Becca Fleischer

Volume 158 Metalloproteins (Part A)

Edited by James F Riordan and Bert L Vallee

Volume 159 Initiation and Termination of Cyclic Nucleotide ActionEdited by Jackie D Corbin and Roger A Johnson

Volume 160 Biomass (Part A: Cellulose and Hemicellulose)

Edited by Willis A Wood and Scott T Kellogg

Volume 161 Biomass (Part B: Lignin, Pectin, and Chitin)

Edited by Willis A Wood and Scott T Kellogg

Volume 162 Immunochemical Techniques (Part L: Chemotaxis

and Inflammation)

Edited by Giovanni Di Sabato

Volume 163 Immunochemical Techniques (Part M: Chemotaxis

and Inflammation)

Edited by Giovanni Di Sabato

Volume 164 Ribosomes

Edited by Harry F Noller, Jr., and Kivie Moldave

Volume 165 Microbial Toxins: Tools for Enzymology

Edited by Sidney Harshman

Volume 166 Branched-Chain Amino Acids

Edited by Robert Harris and John R Sokatch

Volume 167 Cyanobacteria

Edited by Lester Packer and Alexander N Glazer

Volume 168 Hormone Action (Part K: Neuroendocrine Peptides)Edited by P Michael Conn

Volume 169 Platelets: Receptors, Adhesion, Secretion (Part A)

Edited by Jacek Hawiger

Volume 170 Nucleosomes

Edited by Paul M Wassarman and Roger D Kornberg

Volume 171 Biomembranes (Part R: Transport Theory: Cells and ModelMembranes)

Edited by Sidney Fleischer and Becca Fleischer

Volume 172 Biomembranes (Part S: Transport: Membrane Isolation andCharacterization)

Edited by Sidney Fleischer and Becca Fleischer

Volume 173 Biomembranes [Part T: Cellular and Subcellular Transport:Eukaryotic (Nonepithelial) Cells]

Edited by Sidney Fleischer and Becca Fleischer

Volume 174 Biomembranes [Part U: Cellular and Subcellular Transport:Eukaryotic (Nonepithelial) Cells]

Edited by Sidney Fleischer and Becca Fleischer

Volume 175 Cumulative Subject Index Volumes 135–139, 141–167

Volume 176 Nuclear Magnetic Resonance (Part A: Spectral Techniques andDynamics)

Edited by Norman J Oppenheimer and Thomas L James

Volume 177 Nuclear Magnetic Resonance (Part B: Structure and Mechanism)Edited by Norman J Oppenheimer and Thomas L James

Volume 178 Antibodies, Antigens, and Molecular Mimicry

Edited by John J Langone

Volume 179 Complex Carbohydrates (Part F)

Edited by Victor Ginsburg

Volume 180 RNA Processing (Part A: General Methods)

Edited by James E Dahlberg and John N Abelson

Volume 181 RNA Processing (Part B: Specific Methods)

Edited by James E Dahlberg and John N Abelson

Volume 182 Guide to Protein Purification

Edited by Murray P Deutscher

Volume 183 Molecular Evolution: Computer Analysis of Protein and NucleicAcid Sequences

Edited by Russell F Doolittle

Volume 184 Avidin-Biotin Technology

Edited by Meir Wilchek and Edward A Bayer

Volume 185 Gene Expression Technology

Edited by David V Goeddel

Volume 186 Oxygen Radicals in Biological Systems (Part B: Oxygen Radicalsand Antioxidants)

Edited by Lester Packer and Alexander N Glazer

Volume 187 Arachidonate Related Lipid Mediators

Edited by Robert C Murphy and Frank A Fitzpatrick

Volume 188 Hydrocarbons and Methylotrophy

Edited by Mary E Lidstrom

Volume 189 Retinoids (Part A: Molecular and Metabolic Aspects)

Edited by Lester Packer

Volume 190 Retinoids (Part B: Cell Differentiation and Clinical Applications)Edited by Lester Packer

Volume 191 Biomembranes (Part V: Cellular and Subcellular Transport:Epithelial Cells)

Edited by Sidney Fleischer and Becca Fleischer

Volume 192 Biomembranes (Part W: Cellular and Subcellular Transport:Epithelial Cells)

Edited by Sidney Fleischer and Becca Fleischer

Volume 193 Mass Spectrometry

Edited by James A McCloskey

Volume 194 Guide to Yeast Genetics and Molecular Biology

Edited by Christine Guthrie and Gerald R Fink

Volume 195 Adenylyl Cyclase, G Proteins, and Guanylyl CyclaseEdited by Roger A Johnson and Jackie D Corbin

Volume 196 Molecular Motors and the Cytoskeleton

Edited by Richard B Vallee

Volume 197 Phospholipases

Edited by Edward A Dennis

Volume 198 Peptide Growth Factors (Part C)

Edited by David Barnes, J P Mather, and Gordon H Sato

Volume 199 Cumulative Subject Index Volumes 168–174, 176–194Volume 200 Protein Phosphorylation (Part A: Protein Kinases: Assays,Purification, Antibodies, Functional Analysis, Cloning, and Expression)Edited by Tony Hunter and Bartholomew M Sefton

Volume 201 Protein Phosphorylation (Part B: Analysis of ProteinPhosphorylation, Protein Kinase Inhibitors, and Protein Phosphatases)Edited by Tony Hunter and Bartholomew M Sefton

Volume 202 Molecular Design and Modeling: Concepts and Applications(Part A: Proteins, Peptides, and Enzymes)

Edited by John J Langone

Volume 203 Molecular Design and Modeling: Concepts and Applications(Part B: Antibodies and Antigens, Nucleic Acids, Polysaccharides,and Drugs)

Edited by John J Langone

Volume 204 Bacterial Genetic Systems

Edited by Jeffrey H Miller

Volume 205 Metallobiochemistry (Part B: Metallothionein and RelatedMolecules)

Edited by James F Riordan and Bert L Vallee

Volume 206 Cytochrome P450

Edited by Michael R Waterman and Eric F Johnson

Volume 207 Ion Channels

Edited by Bernardo Rudy and Linda E Iverson

Volume 208 Protein–DNA Interactions

Edited by Robert T Sauer

Volume 209 Phospholipid Biosynthesis

Edited by Edward A Dennis and Dennis E Vance

Volume 210 Numerical Computer Methods

Edited by Ludwig Brand and Michael L Johnson

Volume 211 DNA Structures (Part A: Synthesis and Physical Analysis ofDNA)

Edited by David M J Lilley and James E Dahlberg

Volume 212 DNA Structures (Part B: Chemical and Electrophoretic Analysis

of DNA)

Edited by David M J Lilley and James E Dahlberg

Volume 213 Carotenoids (Part A: Chemistry, Separation, Quantitation, andAntioxidation)

Edited by Lester Packer

Volume 214 Carotenoids (Part B: Metabolism, Genetics, and Biosynthesis)Edited by Lester Packer

Volume 215 Platelets: Receptors, Adhesion, Secretion (Part B)

Edited by Jacek J Hawiger

Volume 216 Recombinant DNA (Part G)

Volume 219 Reconstitution of Intracellular Transport

Edited by James E Rothman

Volume 220 Membrane Fusion Techniques (Part A)

Edited by Nejat Du¨zgu¨nes,

Volume 221 Membrane Fusion Techniques (Part B)

Edited by Nejat Du¨zgu¨nes,

Volume 222 Proteolytic Enzymes in Coagulation, Fibrinolysis, and

Complement Activation (Part A: Mammalian Blood Coagulation Factors andInhibitors)

Edited by Laszlo Lorand and Kenneth G Mann

Volume 223 Proteolytic Enzymes in Coagulation, Fibrinolysis, and

Complement Activation (Part B: Complement Activation, Fibrinolysis, andNonmammalian Blood Coagulation Factors)

Edited by Laszlo Lorand and Kenneth G Mann

Volume 224 Molecular Evolution: Producing the Biochemical Data

Edited by Elizabeth Anne Zimmer, Thomas J White, Rebecca L Cann, andAllan C Wilson

Volume 225 Guide to Techniques in Mouse Development

Edited by Paul M Wassarman and Melvin L DePamphilis

Volume 226 Metallobiochemistry (Part C: Spectroscopic and PhysicalMethods for Probing Metal Ion Environments in Metalloenzymes andMetalloproteins)

Edited by James F Riordan and Bert L Vallee

Volume 227 Metallobiochemistry (Part D: Physical and SpectroscopicMethods for Probing Metal Ion Environments in Metalloproteins)

Edited by James F Riordan and Bert L Vallee

Volume 228 Aqueous Two-Phase Systems

Edited by Harry Walter and Go¨te Johansson

Volume 229 Cumulative Subject Index Volumes 195–198, 200–227

Volume 230 Guide to Techniques in Glycobiology

Edited by William J Lennarz and Gerald W Hart

Volume 231 Hemoglobins (Part B: Biochemical and Analytical Methods)Edited by Johannes Everse, Kim D Vandegriff, and Robert M WinslowVolume 232 Hemoglobins (Part C: Biophysical Methods)

Edited by Johannes Everse, Kim D Vandegriff, and Robert M WinslowVolume 233 Oxygen Radicals in Biological Systems (Part C)

Edited by Lester Packer

Volume 234 Oxygen Radicals in Biological Systems (Part D)

Edited by Lester Packer

Volume 235 Bacterial Pathogenesis (Part A: Identification and Regulation ofVirulence Factors)

Edited by Virginia L Clark and Patrik M Bavoil

Volume 236 Bacterial Pathogenesis (Part B: Integration of PathogenicBacteria with Host Cells)

Edited by Virginia L Clark and Patrik M Bavoil

Volume 237 Heterotrimeric G Proteins

Edited by Ravi Iyengar

Volume 238 Heterotrimeric G-Protein Effectors

Edited by Ravi Iyengar

Volume 239 Nuclear Magnetic Resonance (Part C)

Edited by Thomas L James and Norman J Oppenheimer

Volume 240 Numerical Computer Methods (Part B)

Edited by Michael L Johnson and Ludwig Brand

Volume 241 Retroviral Proteases

Edited by Lawrence C Kuo and Jules A Shafer

Volume 242 Neoglycoconjugates (Part A)

Edited by Y C Lee and Reiko T Lee

Volume 243 Inorganic Microbial Sulfur Metabolism

Edited by Harry D Peck, Jr., and Jean LeGall

Volume 244 Proteolytic Enzymes: Serine and Cysteine Peptidases

Edited by Alan J Barrett

Volume 245 Extracellular Matrix Components

Edited by E Ruoslahti and E Engvall

Volume 246 Biochemical Spectroscopy

Edited by Kenneth Sauer

Volume 247 Neoglycoconjugates (Part B: Biomedical Applications)

Edited by Y C Lee and Reiko T Lee

Volume 248 Proteolytic Enzymes: Aspartic and Metallo Peptidases

Edited by Alan J Barrett

Volume 249 Enzyme Kinetics and Mechanism (Part D: Developments inEnzyme Dynamics)

Edited by Daniel L Purich

Volume 250 Lipid Modifications of Proteins

Edited by Patrick J Casey and Janice E Buss

Volume 251 Biothiols (Part A: Monothiols and Dithiols, Protein Thiols, andThiyl Radicals)

Edited by Lester Packer

Volume 252 Biothiols (Part B: Glutathione and Thioredoxin; Thiols in SignalTransduction and Gene Regulation)

Edited by Lester Packer

Volume 253 Adhesion of Microbial Pathogens

Edited by Ron J Doyle and Itzhak Ofek

Volume 254 Oncogene Techniques

Edited by Peter K Vogt and Inder M Verma

Volume 255 Small GTPases and Their Regulators (Part A: Ras Family)Edited by W E Balch, Channing J Der, and Alan Hall

Volume 256 Small GTPases and Their Regulators (Part B: Rho Family)Edited by W E Balch, Channing J Der, and Alan Hall

Volume 257 Small GTPases and Their Regulators (Part C: Proteins Involved

in Transport)

Edited by W E Balch, Channing J Der, and Alan Hall

Volume 258 Redox-Active Amino Acids in Biology

Edited by Judith P Klinman

Volume 259 Energetics of Biological Macromolecules

Edited by Michael L Johnson and Gary K Ackers

Volume 260 Mitochondrial Biogenesis and Genetics (Part A)

Edited by Giuseppe M Attardi and Anne Chomyn

Volume 261 Nuclear Magnetic Resonance and Nucleic Acids

Edited by Thomas L James

Volume 262 DNA Replication

Edited by Judith L Campbell

Volume 263 Plasma Lipoproteins (Part C: Quantitation)

Edited by William A Bradley, Sandra H Gianturco, and Jere P SegrestVolume 264 Mitochondrial Biogenesis and Genetics (Part B)

Edited by Giuseppe M Attardi and Anne Chomyn

Volume 265 Cumulative Subject Index Volumes 228, 230–262

Volume 266 Computer Methods for Macromolecular Sequence AnalysisEdited by Russell F Doolittle

Volume 267 Combinatorial Chemistry

Edited by John N Abelson

Volume 268 Nitric Oxide (Part A: Sources and Detection of NO; NOSynthase)

Edited by Lester Packer

Volume 269 Nitric Oxide (Part B: Physiological and Pathological Processes)Edited by Lester Packer

Volume 270 High Resolution Separation and Analysis of Biological

Macromolecules (Part A: Fundamentals)

Edited by Barry L Karger and William S Hancock

Volume 271 High Resolution Separation and Analysis of Biological

Macromolecules (Part B: Applications)

Edited by Barry L Karger and William S Hancock

Volume 272 Cytochrome P450 (Part B)

Edited by Eric F Johnson and Michael R Waterman

Volume 273 RNA Polymerase and Associated Factors (Part A)

Edited by Sankar Adhya

Volume 274 RNA Polymerase and Associated Factors (Part B)

Edited by Sankar Adhya

Volume 275 Viral Polymerases and Related Proteins

Edited by Lawrence C Kuo, David B Olsen, and Steven S CarrollVolume 276 Macromolecular Crystallography (Part A)

Edited by Charles W Carter, Jr., and Robert M Sweet

Volume 277 Macromolecular Crystallography (Part B)

Edited by Charles W Carter, Jr., and Robert M Sweet

Volume 278 Fluorescence Spectroscopy

Edited by Ludwig Brand and Michael L Johnson

Volume 279 Vitamins and Coenzymes (Part I)

Edited by Donald B McCormick, John W Suttie, and Conrad Wagner

Volume 280 Vitamins and Coenzymes (Part J)

Edited by Donald B McCormick, John W Suttie, and Conrad WagnerVolume 281 Vitamins and Coenzymes (Part K)

Edited by Donald B McCormick, John W Suttie, and Conrad WagnerVolume 282 Vitamins and Coenzymes (Part L)

Edited by Donald B McCormick, John W Suttie, and Conrad WagnerVolume 283 Cell Cycle Control

Edited by William G Dunphy

Volume 284 Lipases (Part A: Biotechnology)

Edited by Byron Rubin and Edward A Dennis

Volume 285 Cumulative Subject Index Volumes 263, 264, 266–284, 286–289Volume 286 Lipases (Part B: Enzyme Characterization and Utilization)Edited by Byron Rubin and Edward A Dennis

Volume 287 Chemokines

Edited by Richard Horuk

Volume 288 Chemokine Receptors

Edited by Richard Horuk

Volume 289 Solid Phase Peptide Synthesis

Edited by Gregg B Fields

Volume 290 Molecular Chaperones

Edited by George H Lorimer and Thomas Baldwin

Volume 291 Caged Compounds

Edited by Gerard Marriott

Volume 292 ABC Transporters: Biochemical, Cellular, and Molecular AspectsEdited by Suresh V Ambudkar and Michael M Gottesman

Volume 293 Ion Channels (Part B)

Edited by P Michael Conn

Volume 294 Ion Channels (Part C)

Edited by P Michael Conn

Volume 295 Energetics of Biological Macromolecules (Part B)

Edited by Gary K Ackers and Michael L Johnson

Volume 296 Neurotransmitter Transporters

Edited by Susan G Amara

Volume 297 Photosynthesis: Molecular Biology of Energy Capture

Edited by Lee McIntosh

Volume 298 Molecular Motors and the Cytoskeleton (Part B)

Edited by Richard B Vallee

Volume 299 Oxidants and Antioxidants (Part A)

Edited by Lester Packer

Volume 300 Oxidants and Antioxidants (Part B)

Edited by Lester Packer

Volume 301 Nitric Oxide: Biological and Antioxidant Activities (Part C)Edited by Lester Packer

Volume 302 Green Fluorescent Protein

Edited by P Michael Conn

Volume 303 cDNA Preparation and Display

Edited by Sherman M Weissman

Volume 304 Chromatin

Edited by Paul M Wassarman and Alan P Wolffe

Volume 305 Bioluminescence and Chemiluminescence (Part C)

Edited by Thomas O Baldwin and Miriam M Ziegler

Volume 306 Expression of Recombinant Genes in Eukaryotic SystemsEdited by Joseph C Glorioso and Martin C Schmidt

Volume 307 Confocal Microscopy

Edited by P Michael Conn

Volume 308 Enzyme Kinetics and Mechanism (Part E: Energetics of EnzymeCatalysis)

Edited by Daniel L Purich and Vern L Schramm

Volume 309 Amyloid, Prions, and Other Protein Aggregates

Edited by Ronald Wetzel

Volume 310 Biofilms

Edited by Ron J Doyle

Volume 311 Sphingolipid Metabolism and Cell Signaling (Part A)

Edited by Alfred H Merrill, Jr., and Yusuf A Hannun

Volume 312 Sphingolipid Metabolism and Cell Signaling (Part B)

Edited by Alfred H Merrill, Jr., and Yusuf A Hannun

Volume 313 Antisense Technology (Part A: General Methods, Methods ofDelivery, and RNA Studies)

Edited by M Ian Phillips

Volume 314 Antisense Technology (Part B: Applications)

Edited by M Ian Phillips

Volume 315 Vertebrate Phototransduction and the Visual Cycle (Part A)Edited by Krzysztof Palczewski

Volume 316 Vertebrate Phototransduction and the Visual Cycle (Part B)Edited by Krzysztof Palczewski

Volume 317 RNA–Ligand Interactions (Part A: Structural Biology Methods)Edited by Daniel W Celander and John N Abelson

Volume 318 RNA–Ligand Interactions (Part B: Molecular Biology Methods)Edited by Daniel W Celander and John N Abelson

Volume 319 Singlet Oxygen, UV-A, and Ozone

Edited by Lester Packer and Helmut Sies

Volume 320 Cumulative Subject Index Volumes 290–319

Volume 321 Numerical Computer Methods (Part C)

Edited by Michael L Johnson and Ludwig Brand

Volume 322 Apoptosis

Edited by John C Reed

Volume 323 Energetics of Biological Macromolecules (Part C)

Edited by Michael L Johnson and Gary K Ackers

Volume 324 Branched-Chain Amino Acids (Part B)

Edited by Robert A Harris and John R Sokatch

Volume 325 Regulators and Effectors of Small GTPases (Part D: Rho Family)Edited by W E Balch, Channing J Der, and Alan Hall

Volume 326 Applications of Chimeric Genes and Hybrid Proteins (Part A:Gene Expression and Protein Purification)

Edited by Jeremy Thorner, Scott D Emr, and John N Abelson

Volume 327 Applications of Chimeric Genes and Hybrid Proteins (Part B:Cell Biology and Physiology)

Edited by Jeremy Thorner, Scott D Emr, and John N Abelson

Volume 328 Applications of Chimeric Genes and Hybrid Proteins (Part C:Protein–Protein Interactions and Genomics)

Edited by Jeremy Thorner, Scott D Emr, and John N Abelson

Volume 329 Regulators and Effectors of Small GTPases (Part E: GTPasesInvolved in Vesicular Traffic)

Edited by W E Balch, Channing J Der, and Alan Hall

Volume 330 Hyperthermophilic Enzymes (Part A)

Edited by Michael W W Adams and Robert M Kelly

Volume 331 Hyperthermophilic Enzymes (Part B)

Edited by Michael W W Adams and Robert M Kelly

Volume 332 Regulators and Effectors of Small GTPases (Part F: Ras Family I)Edited by W E Balch, Channing J Der, and Alan Hall

Volume 333 Regulators and Effectors of Small GTPases (Part G: Ras Family II)Edited by W E Balch, Channing J Der, and Alan Hall

Volume 334 Hyperthermophilic Enzymes (Part C)

Edited by Michael W W Adams and Robert M Kelly

Volume 335 Flavonoids and Other Polyphenols

Edited by Lester Packer

Volume 336 Microbial Growth in Biofilms (Part A: Developmental andMolecular Biological Aspects)

Edited by Ron J Doyle

Volume 337 Microbial Growth in Biofilms (Part B: Special Environments andPhysicochemical Aspects)

Edited by Ron J Doyle

Volume 338 Nuclear Magnetic Resonance of Biological Macromolecules(Part A)

Edited by Thomas L James, Volker Do¨tsch, and Uli Schmitz

Volume 339 Nuclear Magnetic Resonance of Biological Macromolecules(Part B)

Edited by Thomas L James, Volker Do¨tsch, and Uli Schmitz

Volume 340 Drug–Nucleic Acid Interactions

Edited by Jonathan B Chaires and Michael J Waring

Volume 341 Ribonucleases (Part A)

Edited by Allen W Nicholson

Volume 342 Ribonucleases (Part B)

Edited by Allen W Nicholson

Volume 343 G Protein Pathways (Part A: Receptors)

Edited by Ravi Iyengar and John D Hildebrandt

Volume 344 G Protein Pathways (Part B: G Proteins and Their Regulators)Edited by Ravi Iyengar and John D Hildebrandt

Volume 345 G Protein Pathways (Part C: Effector Mechanisms)

Edited by Ravi Iyengar and John D Hildebrandt

Volume 346 Gene Therapy Methods

Edited by M Ian Phillips

Volume 347 Protein Sensors and Reactive Oxygen Species (Part A:

Selenoproteins and Thioredoxin)

Edited by Helmut Sies and Lester Packer

Volume 348 Protein Sensors and Reactive Oxygen Species (Part B: ThiolEnzymes and Proteins)

Edited by Helmut Sies and Lester Packer

Volume 349 Superoxide Dismutase

Edited by Lester Packer

Volume 350 Guide to Yeast Genetics and Molecular and Cell Biology (Part B)Edited by Christine Guthrie and Gerald R Fink

Volume 351 Guide to Yeast Genetics and Molecular and Cell Biology (Part C)Edited by Christine Guthrie and Gerald R Fink

Volume 352 Redox Cell Biology and Genetics (Part A)

Edited by Chandan K Sen and Lester Packer

Volume 353 Redox Cell Biology and Genetics (Part B)

Edited by Chandan K Sen and Lester Packer

Volume 354 Enzyme Kinetics and Mechanisms (Part F: Detection andCharacterization of Enzyme Reaction Intermediates)

Edited by Daniel L Purich

Volume 355 Cumulative Subject Index Volumes 321–354

Volume 356 Laser Capture Microscopy and Microdissection

Edited by P Michael Conn

Volume 357 Cytochrome P450, Part C

Edited by Eric F Johnson and Michael R Waterman

Volume 358 Bacterial Pathogenesis (Part C: Identification, Regulation, andFunction of Virulence Factors)

Edited by Virginia L Clark and Patrik M Bavoil

Volume 359 Nitric Oxide (Part D)

Edited by Enrique Cadenas and Lester Packer

Volume 360 Biophotonics (Part A)

Edited by Gerard Marriott and Ian Parker

Volume 361 Biophotonics (Part B)

Edited by Gerard Marriott and Ian Parker

Volume 362 Recognition of Carbohydrates in Biological Systems (Part A)Edited by Yuan C Lee and Reiko T Lee

Volume 363 Recognition of Carbohydrates in Biological Systems (Part B)Edited by Yuan C Lee and Reiko T Lee

Volume 364 Nuclear Receptors

Edited by David W Russell and David J Mangelsdorf

Volume 365 Differentiation of Embryonic Stem Cells

Edited by Paul M Wassauman and Gordon M Keller

Volume 366 Protein Phosphatases

Edited by Susanne Klumpp and Josef Krieglstein

Volume 367 Liposomes (Part A)

Edited by Nejat Du¨zgu¨nes,

Volume 368 Macromolecular Crystallography (Part C)

Edited by Charles W Carter, Jr., and Robert M Sweet

Volume 369 Combinational Chemistry (Part B)

Edited by Guillermo A Morales and Barry A Bunin

Volume 370 RNA Polymerases and Associated Factors (Part C)

Edited by Sankar L Adhya and Susan Garges

Volume 371 RNA Polymerases and Associated Factors (Part D)Edited by Sankar L Adhya and Susan Garges

Volume 372 Liposomes (Part B)

Edited by Negat Du¨zgu¨nes,

Volume 373 Liposomes (Part C)

Edited by Negat Du¨zgu¨nes,

Volume 374 Macromolecular Crystallography (Part D)

Edited by Charles W Carter, Jr., and Robert W Sweet

Volume 375 Chromatin and Chromatin Remodeling Enzymes (Part A)Edited by C David Allis and Carl Wu

Volume 376 Chromatin and Chromatin Remodeling Enzymes (Part B)Edited by C David Allis and Carl Wu

Volume 377 Chromatin and Chromatin Remodeling Enzymes (Part C)Edited by C David Allis and Carl Wu

Volume 378 Quinones and Quinone Enzymes (Part A)

Edited by Helmut Sies and Lester Packer

Volume 379 Energetics of Biological Macromolecules (Part D)

Edited by Jo M Holt, Michael L Johnson, and Gary K AckersVolume 380 Energetics of Biological Macromolecules (Part E)

Edited by Jo M Holt, Michael L Johnson, and Gary K AckersVolume 381 Oxygen Sensing (in preparation)

Edited by Chandan K Sen and Gregg L Semenza

Volume 382 Quinones and Quinone Enzymes (Part B) (in preparation)Edited by Helmut Sies and Lester Packer

Volume 383 Numerical Computer Methods (Part D) (in preparation)Edited by Ludwig Brand and Michael L Johnson

Volume 384 Numerical Computer Methods (Part E) (in preparation)Edited by Ludwig Brand and Michael L Johnson

Volume 385 Imaging in Biological Research (Part A) (in preparation)Edited by P Michael Conn

Volume 386 Imaging in Biological Research (Part B) (in preparation)Edited by P Michael Conn

[1] Contributions to the Catalytic Efficiency of Enzymes, and the Binding of Ligands to Receptors, from Improvements in Packing within Enzymes

and Receptors

By Dudley H Williams, Elaine Stephens,

Min Zhou, and Rosa Zerella

Introduction

One of the great challenges to twenty-first-century science is to furtherour understanding of the noncovalent interactions that are responsible forthe molecule-to-molecule binding that is the key to biological function.Suppose we were given a picture of a set of noncovalent interactions in-volved in the association of two entities (e.g., from X-ray crystallography)

If we were then able to predict successfully the binding constant (say, towithin a factor of 10), we could claim a relatively good understanding ofnoncovalent interactions Among such attempts, the approach known asLUDI1a is—given its simplicity—moderately successful LUDI builds on

an equation developed in our own laboratory.2 Its modified version1a is

Eq (1)

G¼
Gtþrþ 
Grþ Areað
GhÞ þ 
Ghbþ 
Gionic (1)

In this equation,
G is the observed free energy of a bimolecular ciation Since
G ¼ RT ln K,
G determines the binding constant K.Five common parameters (right-hand side of the equation) that are known

asso-to be important in binding are considered It is assumed that their sum willgive a useful approximation of
G, and hence of K

The first two terms oppose binding
Gtþr is the free energy cost ofrestricting the overall motion of a ligand when it binds to its receptor

Gris the free energy cost of restricting an internal rotation of the ligandthat is restrained upon binding (summed over all such rotations) Boththese terms are essentially adverse entropy terms

The remaining three terms promote binding
Ghis the free energy fit due to the removal of 1 A˚2 of hydrocarbon surface area from waterupon binding (the hydrophobic effect)
Ghis therefore multiplied by the

bene-1 (a) H.-J Bo¨hm, J Comp Aided Mol Des 8, 243 (1994) (b) H.-J Bo¨hm, personal communication (2001).

2 D H Williams, J P L Cox, A J Doig, M Gardner, U Gerhard, P T Kaye, A R Lal,

I A Nicholls, C J Salter, and R C Mitchell, J Am Chem Soc 113, 7020 (1991).

Copyright 2004, Elsevier Inc All rights reserved.

buried surface area for each specified case
Ghbis the free energy benefit

of a hydrogen bond in the binding (summed over all such hydrogen bonds)

Gionic is the free energy benefit of an ionic bond in the binding site(summed over all such ionic bonds)

To ‘‘train’’ the equation, a set of 45 complexes with experimentallyknown binding constants was used In these complexes, ligands of relativelysmall molecular weight (66 to 1047) interact with proteins through sets ofknown interactions (determined by X-ray crystallography) Since Eq (1)has only five types of
G contributions, and the 45 binding sites involvedifferent combinations of these five types of
G contributions, averagevalues for them can be obtained Using these average values, the equationcan then be used to estimate binding constants where ‘‘pictures’’ of bindingsites are available The equation is remarkably successful, for in a limiteddata set (but one that includes compounds outside the training set) it is able

to predict binding constants with a standard deviation of only log101.7.1aHowever, in a wider data set, it performs less well.1bEstimated bindingconstants can be in error by a factor of 1000, or more Partly this is becauseother important terms (e.g., other favorable terms such as
–
stacking) areneglected Partly, it is because cooperativity is neglected Some physicalconsequences of cooperative binding are the subject of this chapter

Cooperativity

Cooperativity is the phenomenon through which one set of bindinginteractions can change the binding energy of another Equation (1)ignores such cooperativity However,Eq (1)shows that sets of interactionsacting simultaneously can give more binding energy than the sum of thesets when occurring separately This point can be understood by reference

toFig 1A and B Suppose that Z can make interactions to its receptor cupthat promote binding by a factor of 103M1 Let the cost of restrictingthe motion of Z into its receptor cup (
Gtþr) oppose binding by a factor

of 102 M1 The binding constant of Z to the receptor would therefore

be 101M1 Let Y, when bound alone, interact with the same parametersinto its (central) receptor cup The binding constant of Y to its receptorcup would therefore also be 101M1

Equation (1)tells us that it would be false to conclude that X–Y (Fig 1B,where X and Y are connected with a strain-free connection, allowing bothgroups to bind in the geometry as when binding separately) would exhibit abinding constant of 101 101¼ 102M1(the sum of the parts).Equation(1) assumes that the cost of a bimolecular association (
Gtþr) has to bepaid only once.2Therefore, the estimated binding constant of X–Y to thereceptor is 103 103/102M1¼ 104M1(greater than the sum of the parts)

The assumption of a useful average
Gtþrterm inEq (1)is an Achillesheel of the approach It implies that all ligands are restricted in motion tothe same, or similar, extents That is, that the degree to which Z isrestrained in Fig 1A is essentially the same as the degree to which Z

is restrained in Fig 1B, i.e., d0 ¼ d1 However, the free energy cost

Gtþr is not a standard cost that is paid for any bimolecular association.Rather, the cost becomes greater as the motions of a ligand relative to itsreceptor become more restricted by stronger bonds (bonds that are formedwith a greater exothermicity).3

Awareness of the above fact points to a problem for any approach tothe estimation of binding constants that treats individual interactions asthough (when formed with the same geometry) they have usefully constant

3 M S Westwell, M S Searle, J Klein, and D H Williams, J Phys Chem 100, 16000 (1996).

Fig 1 Schematic representation of a receptor that binds ligands X, Y, and Z (A) Binding

of Z results in a structure with intermolecular distance d0 (B) When Y and Z are connected

by a rigid, strain-free linker (Y–Z), if they bind the receptor without positive cooperativity, then d0¼ d 1 If they bind with positive cooperativity, there is structural tightening (d1< d0) (C) If X is connected to Y–Z by a rigid, strain-free linker to form X–Y–Z then positively cooperative will cause further structural tightening (d2< d1) (D) The shorter linker between

Y and Z does not allow both these binding interactions to occur with optimal geometries Y–Z binds the receptor with negative cooperativity, and there is structural loosening (d3> d0).

free energy benefits [e.g., for terms 4 and 5 inEq (1)] It is motion that poses bonding In light of this observation, reconsider cases where anumber of noncovalent interactions can be simultaneously made in astrain-free manner to promote the ligand/receptor binding (Fig 1) Themotions about a specified noncovalent interaction (Fig 1A) will typicallybecome more restricted as the ligand is held in place by more adjacentnoncovalent bonds (Fig 1A! Fig 1B ! Fig 1C) The specified inter-action (Fig 1A) then forms with a more favorable enthalpy (i.e., it is asso-ciated with better bonding), but with an increased cost in entropy (a greaterrestriction in motion) Each of the three noncovalent interactions made by

op-X, Y, and Z to a receptor give rise to better bonding when they aremade simultaneously rather than separately Evidence for the effectsmodeled inFig 1A–Cis available from proton nuclear magnetic resonance(NMR) experiments carried out on the binding of ligands to glycopeptideantibiotics,4and is detailed in the following section

Positively Cooperative Binding Probed by NMR Spectroscopy

Several peptide ligands, all containing the carboxyl group depicted atthe lower right in Fig 2, were separately bound to the antibiotics In allcases, a downfield chemical shift of the antibiotic amide NH proton w2was observed upon ligand binding A larger limiting downfield shift of

w2indicates a shorter carboxylate to NH hydrogen bond This hydrogenbond was found to decrease in length as the number of the adjacent hydro-gen bonds that aid ligand binding was increased The motional restriction

of the carboxylate group afforded by these additional hydrogen bondsshortens the hydrogen bonds directly made to the carboxylate.4Analogouseffects have been observed at other interfaces.5

Although the above experiments establish the shortening of lent bonds as a consequence of positive cooperativity, they do not provethat the noncovalent bonds are thereby improved in terms of their freeenergy benefit The proof that the benefit in improved bonding (increasedexothermicity) outweighs the cost in entropy (more restricted motion) isseen in cases in which two interfaces made simultaneously give a largerfree energy of association than the sum of their parts Dimers of glyco-peptide antibiotics of the vancomycin group are further stabilized whenthey bind two molecules of the bacterial cell wall analogues (Fig 3) Thedimeric system is stabilized by the ligand binding, with attendant distance

noncova-4 M S Searle, G J Sharman, P Groves, B Benhamu, D A Beauregard, M S Westwell,

R J Dancer, A J Maguire, A C Try, and D H Williams, J Chem Soc Perkin Trans 1

2781 (1996).

5 C T Calderone and D H Williams, J Am Chem Soc 123, 6262 (2001).

reductions at the dimer interface.6In nine of nine cases, the positive erativity is associated with a benefit in enthalpy; in eight of nine cases, it isassociated with a cost in entropy.7,8

coop-There are large numbers of papers9–12that report changes in receptorstructures upon ligand binding, and clear indications that the receptor can

in some cases be stabilized The antibiotic work indicates some specificcorrelations that increase our understanding:

6 D H Williams, A J Maguire, W Tsuzuki, and M S Westwell, Science 280, 711 (1998).

7 D McPhail and A Cooper, J Chem Soc Faraday Trans 93, 2283 (1997).

8 D H Williams, C T Calderone, and D P O’Brien, J Chem Soc Chem Commun 1266 (2002).

Fig 2 Exploded view of the binding interaction between the glycopeptide antibiotics (in this case vancomycin) and the peptide ligand N--acetyl-Lys-(N-e-acetyl)-d-Ala-d-Ala Hydrogen bonds between the two are indicated by dotted lines The binding is also promoted

by hydrophobic interactions, notably of the Ala methyl groups to the aromatic rings of the antibiotic The amide NH proton W2, mentioned in the text, is labeled.

9 M Gonzalez, L A Bagatolli, I Echabe, J L R Arrondo, C E Argarana, C R Cantor, and G D Fidelio, J Biol Chem 272, 11288 (1997).

10 D C Williams, D C Benjamin, R J Poljak, and G S Rule, J Mol Biol 257, 866 (1996).

11 E Freire, Proc Natl Acad Sci USA 96, 10118 (1999).

12 B A Johnson, E M Wilson, Y Li, D E Moller, R G Smith, and G Zhou, J Mol Biol.

298, 187 (2000).

Fig 3 Peptide backbone of a glycopeptide antibiotic dimer, simultaneously bound to two molecules of a bacterial cell peptide precursor analogue (N-Ac-d-Ala-d-Ala) The binding of the N-Ac-d-Ala-d-Ala occurs with positive cooperativity, such that the dimer system is stabilized and shortens some of the distances at the central (dimer) interface, with an overall benefit in enthalpy and a cost in entropy.

1 The dimeric nature of the receptor system allows the conclusion thattightening (shorter interfacial distances without geometric distor-tion) of an internal interface of the receptor induces increasedstability of the receptor system.

2 The increased stability associated with the positive cooperativity ischaracterized by increased exothermicity and a cost in entropy

3 A thermodynamic cycle establishes that increased stability of thereceptor system when the ligand is bound necessarily leads toincreased ligand-binding energy.13

Binding to Protein Receptors and the Use of Mass SpectrometryFrom the above experiments, we can conclude that where the structure

of a receptor undergoes tightening (reduced internal noncovalent tances) upon ligand binding, ligand binding is thereby enhanced The prop-erties of positively cooperative binding found above for a receptor dimerinterface (Fig 3) are equally applicable when the dimer interface isreplaced by an interface that is within a monomeric receptor (Fig 4) Ineach panel of Fig 4, the ligand is represented as the upper molecule (adipeptide) and the receptor as the lower structure (with illustration ofonly one set of its internal noncovalent interactions, in the form of twoamide–amide hydrogen bonds) The tightening of noncovalent interactions(with exaggerated changes in bond lengths to illustrate the principle)occurs in Fig 4B, where there is positively cooperative binding of ligandthat is absent in Fig 4A Thus, in Fig 4A and B, the proven reductions

dis-in dimer dis-interfacial bond distances upon ligand bdis-inddis-ing with positive operativity are extrapolated to the monomeric receptor case The physicalbasis for the tightening is that the matching fit of the ligand to the exposedbinding site of the receptor causes, through the formation of the ligand/receptor noncovalent bonds, a reduction on the motions of the exposedpart of the receptor (here a peptide backbone) Since it is motion that op-poses bonding, such reductions in motion will be accompanied by bondshortening within the receptor (Fig 4A ! B)

Since the internal tightening of receptor structures upon positively operative ligand binding reduces their dynamic behavior, the extent towhich such tightened receptor structures undergo NH! ND exchange oftheir backbone amide NHs upon exposure to D2O will be decreased Suchchanges in exchange behavior can be conveniently monitored by massspectrometry.14–16 A typical protocol, used to monitor H/D exchange inour laboratory, follows

co-13 B Bardsley and D H Williams, J Chem Soc Chem Commun 2305 (1998).

Fig 4 Schematic representation of a ligand (upper peptide backbone) binding to a receptor (below) (A) in the absence of cooperativity, (B) with positive cooperativity, (C) with negative cooperativity prior to enthalpy/entropy compensation, and (D) with negative

An H/D Exchange Protocol

H/D exchange is typically initiated by dilution of 10 l of a 3 mM tion of a receptor protein in 100 mM ammonium acetate buffer (pH 8.0)into 90 l of 99.9 atom% excess D2O The complex of the receptor proteinwith the appropriate ligand is formed by incubation of 0.3–3 mM receptorwith a 10–20% molar excess of ligand at room temperature for greater than

solu-1 h prior to dilution into D2O Solutions are maintained at room ture for H/D exchange and allowed to exchange for the desired times Atappropriate intervals, 5-l aliquots of the receptor solution are adjusted

tempera-to pH 2.5 by the addition of 30 l of chilled acidic quench solution Thesealiquots are immediately cooled to 0 The use of relatively acidic condi-tions and low temperatures at this stage minimizes the extent of ND !

NH back exchange A-10 l aliquot is then loop injected for electrosprayionization mass spectrometry (ESI-MS) to determine the deuteriumcontent of the receptor system, both in the presence and absence of ligand

Locating the Regions of Structural Tightening in Receptors

The above procedure may indicate the tightening of a receptor systemupon ligand binding and therefore indicates that the ligand-binding energycan be enhanced in this way This was the case found in our laboratory forthe binding of biotin to streptavidin.17Specifically, 22 backbone amide NHprotons per streptavidin are protected from H/D exchange upon biotinbinding Thus, tightening of the streptavidin structure upon the binding

of biotin contributes to the binding affinity of biotin Since the binding ofbiotin occurs to a streptavidin tetramer (which accommodates four mol-ecules of biotin), it is important to determine where the tightening of thestreptavidin tetramer occurs To determine where in the receptor systemthe structural tightening occurs, enzymatic digestion of the receptor iscarried out This can be achieved by two experiments involvingpepsin digestion—of both the ligand-free and ligand-bound receptor Pepsin

14 V Katta and B T Chait, J Am Chem Soc 115, 6317 (1993).

15 (a) Z Zhang and D L Smith, Protein Sci 2, 522 (1993) (b) D L Smith, Y Deng, and

Z Zhang, J Mass Spectrom 32, 135 (1997).

16 F Wang, R W Miles, G Kicsa, E Nieves, V L Schramm, and R H Angeletti, Protein Sci.

9, 1660 (2000).

17 D H Williams, E Stephens, and M Zhou, J Mol Biol 329, 389 (2003).

cooperativity after enthalpy/entropy compensation Where the tightened (B), or loosened (D), interactions are coupled to other interactions within the receptor system, they will be similarly affected.

digestion is used because this enzyme can function at pH 2.5—the pH atwhich back exchange of amide backbone ND! NH is minimized The pep-tide fragments derived from the receptor in both experiments are then ana-lyzed by liquid chromatography (LC)-ESI-MS The relative deuteriumcontents of each set are determined from their molecular weights (in com-parison with those of the corresponding peptides obtained in the absence

of H/D exchange)

The amino acid sequence of the receptor is of course typically known.Therefore, the structures of the peptides produced by pepsin digestioncan be determined from their molecular weights, in combination withsome sequence information derived from their collision-induced frag-mentation A protocol for achieving such digestion, and analyzing theproducts, follows Previously published protocols are available.15,16 Thefollowing protocol was used to show that the peptide backbone NHs thatare protected upon the binding of biotin to streptavidin are widely dis-tributed through the streptavidin.17Thus the binding energy for biotin tostreptavidin is widely delocalized

A Protocol for Pepsin Digestion and Analysis of the Digest (Used in theCase of Streptavidin as the Receptor)

Pepsin digestions can be performed on-line by linking a digestion ridge made by packing a microbore guard column (1 20 mm) (UpchurchScientific) with pepsin Porozyme media (Applied Biosystems) to a Rheo-dyne 7010 injector coupled with LC-MS The protein solution, quenched

cart-to pH 2.5, is injected incart-to the pepsin cartridge and the protein is digestedfor 3 min at 0 The resulting peptide mixture is then infused at 100 lmin1 through a C18 reverse-phase peptide trap for 2 min, using ice-coldbuffer (10 mM ammonium acetate, 2% acetic acid, pH 2.9) When the in-jector is switched to inject mode, the peptide trap is subsequently placedin-line with the LC column (PepMap C18, 300 m  5 cm; LC-Packings,Dionex) and peptides are eluted with increasing organic concentration

An LC-Packings Ultimate capillary high-performance liquid raphy (HPLC) (Dionex) can be used to generate the gradient (flow rate

chromatog-4 l min1), e.g., solvent A 0.1% formic acid in H2O and solvent B 90%acetonitrile containing 9.95% H2O and 0.05% formic acid The peptic pep-tides eluted between 3.5 and 9 min with a 5-min 20–50% B gradient, atwhich time the gradient is held at 50% B for 10 min The column effluent

is delivered directly to a nanoflow ESI probe held at 3 kV For all ments, the solvents, Rheodyne injectors, peptide trap, and HPLC columnare all immersed in an ice bath (0) to minimize back exchange with sol-vents To account for deuterium gain or loss under quenched conditions,

experi-two control samples are prepared A ‘‘zero deuteration’’ control is pared by diluting the protein solution directly into a 1:1 (v:v) mixture ofdeuterated buffer and quench buffer A ‘‘full-deuteration’’ control is pre-pared by incubating streptavidin in 8 M urea-d4 in D2O at 55 for 2 h.Using the above protocol, the extent of deuterium loss in the pepticpeptides was ca 30–50%, consistent with previous reports.16 The deuter-ium content of each peptide can be calculated after correction for backexchange, as described previously.15

pre-Identification of Peptides from Pepsin Digestion

Peptides can be sequenced by LC-MS/MS following pepsin proteolysisunder conditions identical to those used for the deuterium exchangeexperiments, except that D2O is omitted Switching between MS andMS/MS can be achieved with automatic switching triggered by the detec-tion of specific peptide ions entered into Masslynx software as a peak list.Argon is used as the collision gas and collision energies from 32 to 35 eVare typically applied

Evidence that Enzymes Derive Catalytic Efficiency by Tightening TheirStructures to the Greatest Degree in the Transition State

The concept that when a small molecule (L) binds to a protein (P),binding energy of L to P is derived by tightening (contracting) the structure

of P, has potential implications for enzyme catalysis If enzymes exploit thiseffect to derive binding energy of the substrate and product, then theenzyme structure should be contracted when substrate and product arebound However, if enzymes exploit this effect to increase catalytic effi-ciency, then enzyme structures should be contracted to the greatest extent

in the transition state for reaction This last point follows since, for efficientcatalysis, the free energy of the substrate transition state–enzyme systemmust be lowered to the greatest degree

Thermodynamic Evidence for Better Packing of Enzymes in the

Transition States

A reaction [S! P,Eq (2)] catalyzed by an enzyme (E) benefits, tive to the reaction in free solution, because the adverse entropy of thereaction in free solution is reduced by the preorganization of the catalyticgroups in relation to the substrate (S).18Catalysis will also be promoted

rela-18 A Fersht, ‘‘Structure and Mechanism in Protein Science,’’ p 362 W H Freeman and Co., New York, 1999.

if the enzyme binds the transition state (S#) for reaction with positive operativity According to the model presented here, such positivelycooperative binding will give a benefit in enthalpy and a cost in entropydue to better packing within the enzyme structure in the transition state[E S#,Eq (2)] The prediction is therefore that this latter cost in entropywill offset the advantage of the preorganization, but that a large benefit inenthalpy should be apparent in enzyme catalysis.

The extent to which enzyme catalysis is provided by any overall provement in bonding (

H#) is available from the difference betweenthe enthalpy of activation for the enzyme-catalyzed reaction (
H#cat) andfor the spontaneous reaction in the absence of enzyme (
H#non) The cost

im-or benefit to catalysis in terms of an overall change in im-order (T

S#) isavailable from the difference of the parameters T
S#cat and T
S#non forthe same two processes These differences are available for the reactioncatalyzed by cytidine deaminase.19The effect of enzyme catalysis is to in-crease the reaction rate by 1016M1, due to a benefit in enthalpy (

H#)

of 84 kJ mol1, and a benefit in entropy (T

S#) of only 7 kJ mol1.From the Boltzmann equation, 5.7 kJ mol1 benefits a reaction rate

at room temperature by a factor of 101 Thus, the benefit of improvedbonding to the enzyme-catalyzed reaction is a factor of ca 1015, whereasthe benefit due to improved order is only a factor of ca 101 The very largeoverall improvement in bonding in the transition state of the enzyme-catalyzed reaction is consistent with catalysis being derived to a majorextent by a tightening of the enzyme structure, induced by the transitionstate of the substrate

The above data are therefore interpreted to reflect to an importantdegree the increased bonding within the enzyme on passing from its free

to transition-state–bound form The expectation is that much of this creased bonding within the enzyme will be derived on passing from theenzyme/substrate complex to the form that is bound by the transition state

in-of the substrate This is because enzymes have evolved to bind the tion states (S#) of substrates more strongly than the substrates themselves.One way to effect this is through a greater degree of tightening of theenzyme upon binding the substrate transition state than upon binding thesubstrate The enthalpic (bonding) component of this difference for cyti-dine deaminase19is30 kJ mol1, which establishes that there is an overallincrease in bonding as the reaction proceeds from the enzyme-bound

transi-19 M J Snider, S Gaunitz, C Ridgeway, S A Short, and R Wolfenden, Biochemistry 39,

9746 (2000).

ground state to the transition state The data are consistent with animprovement in bonding within the enzyme during this transformation.The efficiency of enzyme catalysis can be measure in terms of the rateratio of the enzyme-catalyzed and non-catalyzed reactions (kcat/knon) Theenthalpic (bonding) component of this difference has been measured byWolfenden and co-workers20,21 for reactions catalyzed by six enzymes.The work establishes that these reactions are accelerated largely as a result

of a more favorable enthalpy of activation (in comparison to the reaction infree solution) These contributions are33 (chorismate dismutase), 66(chymotrypsin), 63 (staphylococcal nuclease), 80 (bacterial -glucosi-dase),93 (urease), and 143 (yeast OMP decarboxylase) kJ mol1 Sincethese differences are derived by comparison of reactions that both involvethe transition state of the substrate, they must largely reflect bondingchanges in the surroundings of this transition state structure They give rise,when considered in isolation from other variables, to rate enhancements of

ca 106, 1012, 1011, 1014, 1016, and 1025 s1, respectively These enthalpychanges are so large that widespread improvements in bonding in theenzyme structures, induced by the transition state of the substrate, offer aprobable explanation

Evidence for Better Packing and Reduced Dynamic Behavior from

Although there is evidence in the literature that some enzymes becomebetter packed in the transition state for reaction, the point that this mustimprove catalytic efficiency has not been made clear The key data comefrom experiments carried out by Wang et al.16,22 Hydrogen/deuterium(H/D) exchange into backbone amide bonds in hypoxanthine-guaninephosphoribosyltransferase (HGPRT)22 and purine nucleoside phosphory-lase16was used to compare the dynamic properties of the enzymes alone,

in forms with bound reactant/product, and in forms with bound tion-state analogues For both enzymes, it was found that the rate andextent of deuterium incorporation decreased when the reactant/productwas bound, and decreased to an even greater extent when the transitionstate analogue was bound Thus, the greatest reduction in dynamic motion

transi-of the enzymes is caused by the transition state analogue The effects arelarge: the binding of the transition state analogue protects 34 peptide back-bone NHs from exchange in the case of HGPRT, and 27 peptide backbone

20 A Radzicka and R Wolfenden, Science 267, 90 (1995).

21 R Wolfenden, M Snider, C Ridgway, and B Miller, J Am Chem Soc 121, 7419 (1999).

22 F Wang, W Shi, E Nieves, R H Angeletti, V L Schramm, and C Grubmeyer, Biochemistry 40, 8043 (2001).

NHs are similarly protected in the case of purine nucleoside phosphorylase.The reduced dynamic behavior of the enzymes goes hand in hand with im-proved noncovalent bonding within them Our proposals indicate that forboth enzymes binding energy is provided for the reactant/product throughthe enzymes becoming better packed More importantly, even greaterbinding energy is provided for the transition state analogue when theenzyme packing is further improved.

Negatively Cooperative Binding of Ligands and Structural

Loosening in Receptors

So far, we have presented the case that positively cooperative bindingcan cause the tightening of noncovalently bonded structures Negativelycooperative binding is the converse of positively cooperative binding.Therefore, it should be associated with converse properties, i.e., a reduc-tion of the noncovalent bonding efficiency within the receptor system,and an increase in its dynamic behavior These consequences of negativelycooperative binding should occur with a cost in enthalpy and a benefit inentropy

The physical model for an increase in receptor dynamics upon the cise of negative cooperativity involves arguing via two hypothetically sep-arated steps The first step is that the ligand binds by making noncovalentbonds to the receptor whose formation demands distortion of the noncova-lent bonds that previously existed within the receptor That is, making si-multaneously the two sets of bonds in the preferred geometry that wouldoccur if each set were made alone is not possible Thus, the making ofthe ligand/receptor bonds decreases the bonding efficiency of the noncova-lent bonds within the receptor (Fig 4C)—there has been a cost in enthalpy

exer-In the second step, we consider the dynamic consequence of this cost inenthalpy The decrease in bonding within the receptor will result in anincrease in its dynamic behavior, which will in turn cause a further cost

in enthalpy (Fig 4C ! D)

We were encouraged that the model might have general applicability by

a study of changes in the properties of tetrameric recombinant human sine hydroxylase isoform 1 upon binding the natural cofactor (6R)-l-erythro-5,6,7,8-tetrahydrobioptrin.23 The binding of the cofactor occurs withnegative cooperativity, and this cofactor-bound form of the enzyme thenshows a decreased resistance to limited tryptic proteolysis—as would beexpected from a loosening of the enzyme structure

tyro-23 T Flatmark, B Almas, P M Knappskog, S V Berge, R M Svebak, R Chehin, A Muga, and A Martinez, Eur J Biochem 262, 840 (1999).